CN107854544B - Traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress and preparation method and application thereof - Google Patents
Traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress, which is prepared from the following components in parts by weight: 20-60 parts of echinacea purpurea, 20-60 parts of scutellaria baicalensis, 10-50 parts of agastache rugosus, 10-50 parts of elsholtzia, 8-35 parts of gypsum, 8-35 parts of pericarpium citri reticulatae, 3-25 parts of bighead atractylodes rhizome and 3-25 parts of liquorice. The invention also discloses a preparation method and application of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress. The invention has the following advantages and beneficial effects: the injury of the heat stress to the chicken can be relieved by influencing the biochemical indexes of the heat stress chicken blood and adjusting the intestinal flora of the caecum.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the field of traditional Chinese veterinary medicines, in particular to a traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress and a preparation method thereof.
[ background of the invention ]
Heat stress is all non-specific physiological reactions generated when the body faces high temperature environment. The heat stress can reduce the food intake and water intake of the organism, reduce the production performance, cause the continuous ischemia of the gastrointestinal tract, disorder of intestinal flora, digestive dysfunction and the like. Intestinal microorganisms are the largest and most important micro-ecosystem of the animal body, and the intestinal tract plays a crucial role in certain disease processes, for example, the intestinal tract is the initiator of sepsis multiple organ dysfunction syndrome. Under the condition of heat stress, the chicken intestinal tract is easy to suffer from ischemia and anoxic damage, the top of epithelial villus falls off to form ulcer, the intestinal permeability is increased, and the like.
[ summary of the invention ]
The invention aims to overcome the defects in the prior art and provides a traditional Chinese medicine composition capable of clearing heat and purging fire, relieving summer heat and eliminating dampness, regulating qi and strengthening spleen, and regulating heat stress chicken intestinal flora under heat stress by regulating heat stress chicken blood biochemistry and intestinal flora.
The invention also aims to provide a preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress.
The third purpose of the invention is to provide the application of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress.
In order to achieve the above object, the present invention is realized by: a traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 20-60 parts of echinacea purpurea, 20-60 parts of scutellaria baicalensis, 10-50 parts of agastache rugosus, 10-50 parts of elsholtzia, 8-35 parts of gypsum, 8-35 parts of pericarpium citri reticulatae, 3-25 parts of bighead atractylodes rhizome and 3-25 parts of liquorice.
Preferably, the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 40 parts of echinacea purpurea, 40 parts of scutellaria baicalensis, 30 parts of agastache rugosus, 30 parts of elsholtzia, 20 parts of gypsum, 20 parts of dried orange peel, 10 parts of bighead atractylodes rhizome and 10 parts of liquorice.
A preparation method of a traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress comprises the following steps:
(1) soaking herba Moslae, herba Agastaches, and pericarpium Citri Tangerinae in water, and extracting volatile oil;
(2) adding the medicine residues obtained in the step (1) into a pot, and decocting the medicine residues with echinacea, scutellaria baicalensis, gypsum, bighead atractylodes rhizome and liquorice with water;
(3) concentrating the water decoction obtained in the step (2) to a density of 0.5-1.5 g/mL;
(4) dissolving the volatile oil obtained in the step (1) by using Tween-80, then uniformly mixing with the concentrated solution obtained in the step (3), and adding potassium sorbate with the total mass of 0.1-0.5% for preservation to prepare decoction. Preferably, the addition amount of the potassium sorbate is 0.3%.
In the step (1), the soaking time is 1h, and the volatile oil is extracted by a steam distillation method, and can be directly heated and distilled or distilled by introducing steam.
The distillation time for extracting the volatile oil by steam distillation is 8 hours, so that the volatile oil in the medicinal materials can be effectively extracted.
In the step (2), water is added in an amount which is 5-12 times of the weight of the medicinal materials, gypsum is decocted for 1-3h for 15-45min, and the decoction is repeated for 1-5 times. Preferably, water is added in an amount which is 8 times of the weight of the medicinal materials, gypsum is decocted for 2 hours firstly and then decocted together, and the decoction is repeated for 3 times.
In the step (3), the water decoction obtained in the step 2) is concentrated by using a rotary evaporator.
The weight ratio of the volatile oil to the Tween-80 in the step (4) is 1:1-3, preferably, the weight ratio of the volatile oil to the Tween-80 is 1: 2.
The application of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is that the addition amount of the traditional Chinese medicine composition in chicken drinking water is 0.1-1%. Preferably, the addition amount of the additive in the drinking water for chicken is 0.25 percent, or the addition amount of the additive in the drinking water for chicken is 0.5 percent.
The theory of traditional Chinese medicine considers that heat stress is mainly caused by excess heat in qi system, and then develops into nutrient heat and blood heat. The traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress disclosed by the invention is tested on the influence of the chicken intestinal flora under heat stress, and the test result shows that the serum ALT, AST, TP, ALB and GLU of a high-temperature group are remarkably changed (P is less than 0.05) compared with a moderate-temperature group at 7d (day), and the difference between the traditional Chinese medicine and the moderate-temperature group is not remarkable (P is more than 0.05); the high-temperature group can obviously reduce the abundance and diversity of chicken cecal intestinal flora, and the traditional Chinese medicine group can obviously increase the abundance and diversity (P is less than 0.05); the abundance ratio of caecum and cepiteophyceae of the chickens is obviously different from that of the thermophilic group (P is less than 0.05) under the condition of heat stress, and the change of the caecum and cepiteophyceae of the chickens can be improved by the traditional Chinese medicine group. The results show that the traditional Chinese medicine compound decoction can relieve the damage of heat stress to the chickens by influencing the biochemical indexes of the blood of the heat stress chickens and adjusting the intestinal flora of caecum.
Compared with the prior art, the invention has the following advantages and beneficial effects: the injury of the heat stress to the chicken can be relieved by influencing the biochemical indexes of the heat stress chicken blood and adjusting the intestinal flora of the caecum.
[ description of the drawings ]
FIG. 1 is an observed _ species curve;
FIG. 2 is a Shannon curve;
FIG. 3 is a plot of chao 1;
FIG. 4 is a Rank-abundance curve;
FIG. 5 is a plot of the weighted PCoA analysis of trial 1 d;
FIG. 6 is a plot of the unweighted PCoA analysis of trial 1 d;
FIG. 7 is a graph of the test 3d weighted PCoA analysis;
FIG. 8 is a plot of the unweighted PCoA analysis of trial 3 d;
FIG. 9 is a plot of the test 7d weighted PCoA analysis;
FIG. 10 run 7d unweighted PCoA analysis chart;
FIG. 11, run 14d weighted PCoA analysis;
FIG. 12 run 14d unweighted PCoA analysis chart;
FIG. 13 plot of run 28d weighted PCoA analysis;
figure 14 run 28d unweighted PCoA analysis plot.
[ detailed description ] embodiments
The present invention will be described in detail with reference to specific examples.
Example 1
A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 21 parts of echinacea purpurea, 21 parts of scutellaria baicalensis, 11 parts of agastache rugosus, 11 parts of elsholtzia, 9 parts of gypsum, 9 parts of pericarpium citri reticulatae, 4 parts of bighead atractylodes rhizome and 4 parts of liquorice.
Example 2
A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 59 parts of echinacea, 59 parts of scutellaria baicalensis, 49 parts of agastache rugosus, 49 parts of elsholtzia, 34 parts of gypsum, 34 parts of dried orange peel, 24 parts of bighead atractylodes rhizome and 24 parts of liquorice.
Example 3
A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 30-50 parts of echinacea purpurea, 30-50 parts of scutellaria baicalensis, 20-40 parts of agastache rugosus, 20-40 parts of elsholtzia, 12-20 parts of gypsum, 12-20 parts of pericarpium citri reticulatae, 5-15 parts of bighead atractylodes rhizome and 5-15 parts of liquorice.
Example 4
A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 35 parts of echinacea purpurea, 45 parts of scutellaria baicalensis, 28 parts of agastache rugosus, 30 parts of elsholtzia, 15 parts of gypsum, 15 parts of dried orange peel, 6 parts of bighead atractylodes rhizome and 10 parts of liquorice.
In the formula, the parts of the traditional Chinese medicine composition are calculated by the weight of crude drugs of traditional Chinese medicinal materials, and can be in g.
The preparation method of the traditional Chinese medicine composition for regulating the chicken intestinal flora under heat stress comprises the following steps:
(1) soaking herba Moslae, herba Agastaches, and pericarpium Citri Tangerinae in water for 1 hr, directly heating and distilling or introducing steam to distill and extract volatile oil, wherein the distillation time is 8 hr;
(2) adding the medicine residues obtained in the step (1) into a pot, decocting the medicine residues, echinacea purpurea, scutellaria baicalensis, gypsum, bighead atractylodes rhizome and liquorice with water, wherein the weight of the water is 8 times of that of the medicinal materials, the gypsum is decocted for 30min, then the gypsum and the water are decocted together for 2h, and the decoction is repeated for three times.
(3) Concentrating the water decoction obtained in the step (2) by using a rotary evaporator until the density is 1 g/mL;
(4) dissolving the volatile oil obtained in the step (1) by using Tween-80, then uniformly mixing with the concentrated solution obtained in the step (3), and adding 0.3% of potassium sorbate to prepare a decoction.
Example 5
A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is prepared from the following components in parts by weight: 40 parts of echinacea purpurea, 40 parts of scutellaria baicalensis, 30 parts of agastache rugosus, 30 parts of elsholtzia, 20 parts of gypsum, 20 parts of dried orange peel, 10 parts of bighead atractylodes rhizome and 10 parts of liquorice.
The preparation method comprises the following steps: soaking herba Moslae, herba Agastaches, and pericarpium Citri Tangerinae for 1 hr, and steam distilling for 8 hr to extract volatile oil; adding Echinacea purpurea, Scutellariae radix, Gypsum Fibrosum, Atractylodis rhizoma, and Glycyrrhrizae radix into the obtained residue, decocting, adding water 8 times of the medicinal materials, decocting Gypsum Fibrosum for 30min, decocting for 2 hr, and decocting for three times; concentrating the obtained water decoction by using a rotary evaporator, and finally concentrating to g/mL; dissolving the volatile oil with tween-80 at a ratio of 1:2, mixing with the concentrated solution, adding 0.3% potassium sorbate, and making into decoction.
Effect testing experiment
Clinical tests were conducted using the Chinese medicinal composition obtained in claim 5 of the present invention, and commercial generation of 1-day-old broiler chickens from south-China-female-yellow-meat-chickens from Guangdong Zhiwei agricultural science and technology, Inc.
All the traditional Chinese medicines of echinacea, astragalus, wrinkled gianthyssop, elsholtzia, gypsum, dried orange peel, white atractylodes rhizome, liquorice and the like are purchased from Likantang pharmacy of Guangzhou city; tiangen fecal genome extraction kit, batch number: DP 328-021.
In the test, commercial generation fast large south yellow female dorkings of 1 day old are selected to be fed to 14 days old, 180-feather dorkings with healthy body and uniform body weight are selected and randomly divided into 4 groups, and each group has 45 feathers. Respectively setting as follows: a temperature-suitable group, a traditional Chinese medicine group and a heat stress group. The broiler chickens are raised on the net flatly, the artificial continuous illumination system is adopted, the chicken house is naturally ventilated, and the broiler chickens can eat and drink water freely. The chicken coop was cleaned and disinfected during the test according to the conventional procedure. The test period is 28 days, the basic daily ration fed by each group is the same, and the drinking water treatment and grouping conditions are shown in table 1.
Table 1 experimental design and grouping
6 chickens were randomly selected and killed in each group at 1d, 3d, 7d, 14d, and 28d of the experiment, and about 5g of the cecum content was taken and stored in a refrigerator at-20 ℃ for total DNA extraction and 16S rDNA sequencing. Extracting DNA of the cecal content bacteria by using a DNA extraction kit, detecting the extracted genome DNA by using 1% agarose gel electrophoresis, amplifying by using a 16s rDNA V3-V4 region after all the extracted genome DNA is qualified, sequencing by using an Illumina Miseq sequencer, and finally analyzing biological information.
SPSS19.0 statistical analysis software is used for analyzing the significance of the difference among the groups by single-factor variance, multiple comparison of mean values among the groups is carried out by Duncan's new complex pole difference test method, and the result is ' mean value plus or minus standard deviation '
Where sample n is 6. The histogram is made with GraphPad Prism 5.
As shown in FIGS. 1 to 4, it can be seen from the observed _ speces curves that when the sequencing depth is small, the number of OTUs changes dramatically, the number of OTUs increases greatly with the increase of the sequencing depth, and when the sequencing depth reaches 450000reads, the dilution curve still has an upward trend, indicating that new bacteria still exist in the chicken intestinal tract and are not discovered. When the sequencing depth reaches 50000reads, the Shannon curve reaches a saturation state, which shows that even if the sequencing concentration is increased, only the abundance of the species can be increased, but the diversity cannot be increased, namely the current sequencing amount is enough to carry out the microbial diversity analysis of the sample. FIG. 3 is a plot based on the chao1 index for predicting the total number of species in a microorganism, from which it can be seen that the sample curve is substantially flat, again demonstrating that the sample sequencing amount substantially reflects the abundance of the sample species. In addition, in order to test the abundance and uniformity of intestinal flora species in the sample. Sorting the OTU abundance of each sample, and then drawing a Rank-abundance graph of the relative abundance ratio of the OTU, as shown in FIG. 4, wherein the abundance distribution curve has a large fluctuation range at the beginning, but is in a flat state after the 1500 abundance value, which indicates that the overall trend of the species distribution in the sample is uniform.
As shown in fig. 5 and 6, in experiment 1d, the first principal component and the second principal component have contributions of 29.13%, 21.59%, 9.58% and 7.91% respectively based on weighted and unweighted principal coordinate analysis of UniFrac. As can be seen from the figure, the samples of each group have certain overlapping phenomena, which indicates that the individual difference between the groups is relatively large. As shown in fig. 7 and 8, in experiment 3d, PCoA in each group intersected, but some groups tended to cluster. In the weighted and unweighted coordinate analysis, the heat stress group starts clustering; clustering the two groups of traditional Chinese medicines in the weighted analysis; the difference in the temperature-adaptive group is larger. Indicating that environmental heat stress begins to have an effect on the intestinal flora. As shown in fig. 9 and 10, the clustering trend was evident for each group in trial 7 d. The hyperthermia group was separated from the other three groups in the weighted and unweighted coordinate analysis, and the heat stress group was more dispersed than the traditional Chinese medicine group and the traditional Chinese medicine group in the unweighted group. It is shown that in test 7d, the difference between the intestinal flora and the thermophilic group is caused by heat stress, and the individual difference in the heat stress group is larger than that in the other three groups. The heat stress factor is shown to lead the difference of intestinal flora, and the influence is larger than that of one group and two groups of traditional Chinese medicines. As shown in fig. 11 and 12, in experiment 14d, under the weighting condition, the intestinal flora adjusted by the traditional Chinese medicine starts to approach to the suitable temperature group, which indicates that the traditional Chinese medicine has good adjustment on the intestinal flora. If the weight is not weighted (only considering the species evolution relationship), under the condition that the second principal component and the third principal component respectively contribute 7.87% and 6.99%, the thermophilic group, the traditional Chinese medicine group and the two groups are completely separated from the heat stress group, the groups are respectively gathered in different areas, and the difference in the groups is reduced. As shown in fig. 13 and 14, in the test 28d, in the weighted coordinate analysis, the first coordinate and the third coordinate respectively contribute 9.90% and 33.43%, the thermophilic group is still far away from other groups, the distribution distance between the traditional Chinese medicine and the heat stress component is also obvious, and the two groups of traditional Chinese medicine are widely distributed and are close to both the group of traditional Chinese medicine and the thermophilic group of traditional Chinese medicine. In the unweighted analysis, the heat stress group is more obviously dispersed with the other three groups, and the traditional Chinese medicine group are closer to each other and are all close to the group with the proper temperature. The results show that the intestinal flora aberration caused by heat stress is more and more obvious through principal component analysis along with the increase of heat stress time, the intestinal flora of the heat stress group is obviously changed at 7 th after the heat stress starts compared with the thermophilic group, the difference in the heat stress group is larger than that of other three groups, the change of the heat stress flora caused by the addition of the traditional Chinese medicines tends to approach the thermophilic group, the effect is obvious at 14d and 28d, and the effect is more and more obvious along with the continuous medication of the heat stress.
As shown in tables 2-7 below, from the whole experimental stage, with the increase of broiler chickens, the abundance ratio of firmicutes is increased, and the long-term chronic heat stress can increase the abundance ratio of firmicutes. The abundance ratio of bacteroidetes is in a decreasing trend, and the abundance ratio of bacteroides can be reduced by long-term chronic heat stress; the addition of traditional Chinese medicines in drinking water can increase the bacteroidetes content to a certain degree, and one group of traditional Chinese medicines is superior to two groups of traditional Chinese medicines. In the test, the ratio of each group of proteobacteria fluctuates greatly, the ratio shows a trend of increasing and then reducing in time, the abundance ratio is relatively large in 7d, and the traditional Chinese medicine compound has a trend of increasing the proteobacteria. The traditional Chinese medicine can effectively regulate the reduction of bacteroides caused by heat stress. Heat stress reduces the abundance ratio of oscillatoria, while traditional Chinese medicines tend to increase. In a high-temperature environment, the clostridium is reduced, and the abundance ratio of the clostridium is increased after the traditional Chinese medicine compound is added.
TABLE 2 influence of Chinese herbs on the abundance ratio (%) of Bischofia gallinarum under heat stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
TABLE 3 influence of Chinese herbs on the abundance ratio (%) of Bacteroides gallinarum under thermal stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
TABLE 4 influence of Chinese herbs on abundance ratio (%) of chanterobacteria in chicken intestinal tract under heat stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
TABLE 5 ratio (%) change of abundance of Chinese herbs to Bacteroides under thermal stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
TABLE 6 abundance ratio (%) change of Chinese herbs to Oscillatoria under heat stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
TABLE 7 abundance ratio (%) change of Chinese herbs to delicate genus under heat stress
The data in the same column are marked with different lower case letters to show significant difference (P <0.05), different upper case letters to show significant difference (P <0.01), and letters with the same or without letters to show no significant difference (P > 0.05).
Clinical tests are carried out on the influence of the composition on the intestinal flora of the chickens under heat stress, and results show that the species abundance, the total species number and the diversity of the intestinal flora of the chickens are increased along with the increase of the age of the chickens in days. And the species abundance, the total species number and the diversity of the intestinal flora are increased due to long-term heat stress. From PCoA, the composition in the intestinal tract of the heat stress group is completely separated from the temperature-suitable group along with the increase of the heat stress time, and the addition of the traditional Chinese medicine tends to draw the temperature-suitable group close. The long-term heat stress can increase the abundance ratio of firmicutes and decrease the abundance ratio of bacteroidetes and proteobacteria, and the changes can be changed after the traditional Chinese medicine is used. The heat stress causes the reduction of the abundance ratio of the bacteroides, the clostridium flexneri and the oscillatoria, and can be relieved by the traditional Chinese medicine composition.
Other embodiments of the invention also have similar experimental effects.
While the preferred embodiments of the present invention have been illustrated and described in detail, it should be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings without inventive faculty. Therefore, any technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the present inventive concept should be within the scope of protection defined by the present claims.
Claims (7)
1. A traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress is characterized by being prepared from the following components in parts by weight: 30-50 parts of echinacea purpurea, 30-50 parts of scutellaria baicalensis, 20-40 parts of agastache rugosus, 20-40 parts of elsholtzia, 12-20 parts of gypsum, 12-20 parts of pericarpium citri reticulatae, 5-15 parts of bighead atractylodes rhizome and 5-15 parts of liquorice.
2. The traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress as claimed in claim 1, which is prepared from the following components in parts by weight: 40 parts of echinacea purpurea, 40 parts of scutellaria baicalensis, 30 parts of agastache rugosus, 30 parts of elsholtzia, 20 parts of gypsum, 20 parts of dried orange peel, 10 parts of bighead atractylodes rhizome and 10 parts of liquorice.
3. The preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress according to claim 1, which comprises the following steps:
(1) soaking herba Moslae, herba Agastaches, and pericarpium Citri Tangerinae in water, and extracting volatile oil;
(2) adding the medicine residues obtained in the step (1) into a pot, and decocting the medicine residues with echinacea, scutellaria baicalensis, gypsum, bighead atractylodes rhizome and liquorice with water;
(3) concentrating the water decoction obtained in the step (2) to a density of 0.5-1.5 g/mL;
(4) dissolving the volatile oil obtained in the step (1) by using Tween-80, and then uniformly mixing with the concentrated solution obtained in the step (3).
4. The preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress as claimed in claim 3, wherein in the step (1), the soaking time is 1h, and the extraction of the volatile oil adopts a steam distillation method.
5. The preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress as claimed in claim 4, wherein the distillation time for extracting the volatile oil by steam distillation is 8 h.
6. The preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress as claimed in claim 3, wherein in the step (2), water is added in an amount which is 5-12 times of the weight of the medicinal materials, gypsum is decocted for 30min, then decocted together for 1-3h, and the decoction is repeated for 1-5 times.
7. The preparation method of the traditional Chinese medicine composition for regulating chicken intestinal flora under heat stress according to claim 3, wherein in the step (3), the water decoction obtained in the step (2) is concentrated by using a rotary evaporator.
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